The innate immune response is the cell's first line of defense against virus infection. This response is typified by the induction of interferon, which in turn induces a cascade of gene expression resulting in the production of proteins that play various roles in eliminating the virus. Many viruses, including NIAID Category A, B, and C agents, are adept at counteracting this response, although the mechanisms by which this is accomplished are not well defined. To gain a better understanding of the signaling pathways involved in innate immunity, and the viral mechanisms used to subvert this response, we propose to identify the composition of protein complexes associated with viral proteins that have a demonstrated ability to act as interferon antagonists. These proteins will include the influenza virus NS 1 protein from the type A Texas/36/91 strain, the 1918 pandemic strain, and the avian Hong Kong strain, and the Ebola-Zaire, Ebola- Reston, and Marburg virus VP35 proteins. The NS 1 and VP35 proteins will be tagged using the tandem affinity purification (TAP) system and expressed by transient or stable transfection of appropriate cell lines (A549 human lung epithelial cells for NS1 and Huh7 human liver cells for VP35) in the presence or absence of infection with delNS 1 influenza virus. As an alternative approach, the tagged NS 1 gene will be introduced into a recombinant influenza virus in place of the wild-type gene. Tandem affinity purification will be used to purify protein complexes associated with the tagged proteins, and mass spectrometry and database search algorithms will be used to identify the individual components of the purified complexes. These studies will increase our understanding of the mechanisms used by viruses identified as potential bioterrorism and emerging infectious disease agents to evade the innate immune response and may suggest novel targets for therapeutic intervention.